The present invention relates to a photo-reactive matrix for matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This photo-reactive matrix allows the determination of the oxidation products of probe molecules and of the products of successive reactions involving the oxidation products of the probe molecules. For example, it provides a very efficient method to carry out photo-redox-induced tagging reactions on sample molecules during the MALDI ionization process.
MALDI ionization is a standard ionization technique to transfer globally neutral solid-state samples, in particular containing biomolecules, to gas-phase ions for further analysis by a mass spectrometer. MALDI ionization is such a general ionization technique that it has been applied to a wide range of biomolecules such as peptides and proteins, DNA [G. Corona and G. Toffoli, Comb. Chem. High Throughput Screen., 7 (2004) 707; C. Jurinke, P. Oeth and D. Van Den Boom, App. Biochem. Biotechnol. B, 26 (2004) 147; J. Ragoussis, G. P. Elvidge, K. Kaur and S. Colella, PLoS Genetics, 2 (2006) 0920], glycans and glycoconjugates [D. J. Harvey, Mass Spectrom. Rev., 18 (1999) 349; D. J. Harvey, Proteomics, 5 (2005) 1774; D. J. Harvey, Mass Spectrom. Rev., 25 (2006) 595], lipids [M. Pulfer and R. C. Murphy, Mass Spec. Rev., 22 (2003) 332; J. Schiller, J. Arnhold, S. Benard, M. Muller, S. Reichl and K. Arnold, Anal. Biochem., 267 (1999) 46] and coupled to various types of mass analyzers, such as ion traps (IT), time-of-flight (TOF), quadrupole-time-of-flight (Q-TOF), Fourier-transform Ion Cyclotron Resonance (FT-ICR).
The principle of MALDI ionization lies in the absorption of laser energy by an acidic crystalline matrix mixed with the sample to be analyzed. Upon energy absorption by the matrix, both matrix and analyte molecules are desorbed from the MALDI plate, and charge transfer reactions occur in the MALDI plume, which finally leads to gas-phase analyte ions that can be analyzed by the mass spectrometer [R. Knochenmuss, Analyst, 131 (2006) 966].
Several methods have been designed for MALDI plate preparation. First, different matrix chemicals can be used, such as α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 2,5-dihydroxybenzoic acid (DHB) or 2-(4-hydroxyphenylazo)-benzoic acid (HABA). Second, different matrix deposition methods are available: the simple so-called dried-droplet technique, in which liquid matrix and sample are mixed, a drop of which is deposited on a metallic MALDI plate. Upon liquid evaporation the matrix co-crystallizes with the analyte. Alternatively, the overlayer method consists in depositing first a matrix layer on the MALDI plate, evaporate it, and then deposit a mixture of matrix and analyte over the first matrix layer. The overlayer method usually results in better spot reproducibility and potential flexibility about the choice of solvent used for the second layer crystallization. Several variations of these two methods have been introduced, but all suffer from the same caveats: the liquid evaporation that is necessary for matrix crystallization is poorly controlled and usually results in highly inhomogeneous spots. When the laser beam is focused on particular zones of the same spot, the probed microenvironments can be very different. Moreover, if the liquid sample/matrix mixtures are deposited directly on metallic plates that are usually hydrophilic, the liquid wets the surface and the droplet spills over a large area, which diminishes the final surface concentration of the matrix/analyte mixture.
Several alternative plates/matrices have been introduced over the recent years whether to alleviate the drawbacks listed above, or to add additional functions to the MALDI plates. In the first category, metallic plates covered with patterns of hydrophilic/hydrophobic zones have been proposed to help in confining matrix/analyte mixtures when deposited on the MALDI plate [H. Thomas, J. Havlis, J. Peychl and A. Shevchenko, Rapid Commun. Mass Spectrom., 18 (2004) 923; H. Wei, S. L. Dean, M. C. Parkin, K. Nolkrantz, J. P. O'Callaghan and R. T. Kennedy, J. Mass Spectrom., 40 (2005) 1338; T. Wenzel, K. Sparbier, T. Mieruch and M. Kostrzewa, Rapid Commun. Mass Spectrom., 20 (2006) 785; Y. C. Wu, C. H. Hsieh and M. F. Tam, Rapid. Commun. Mass Spectrom., 20 (2006) 309]; in the second category, plates covered with specific solid-phases presenting different affinities for targeted biomolecules: for example, Cyphergen has introduced polymer-coated MALDI plates that present different affinities for proteins, based on ion exchange and reverse-phase mechanisms. When the different surfaces are exposed to the sample, different proteins adsorb to different surfaces; non-retained proteins and co-solvents can be washed out [G. L. Wright, L. H. Cazares, S. M. Leung, S. Nasim, B. L. Adam, T. T. Yip, P. F. Schellhammer, L. Gong and A. Vlahou, Prost. Cancer Prost. Diseases, 2 (1999) 264]. Due to the intrinsic properties of the polymer matrices used, a MALDI laser can be directly shot on the polymeric surface, resulting in retained-analyte desorption and ionization. Alternatively, such MALDI plates can be derivatized with particular antibodies to capture specific proteins from complex samples, and further analyze them by mass spectrometry. This approach has been introduced by Cyphergen as well as Intrinsic Bioprobes [U. A. Kiernan, K. A. Tubbs, K. Gruber, D. Nedelkov, E. E. Niederkofler, P. Williams and R. W. Nelson, Anal. Biochem., 301 (2002) 49; D. Nedelkov and R. W. Nelson, Anal. Chim. Acta, 423 (2000) 1; R. W. Nelson, D. Nedelkov and K. A. Tubbs, Anal. Chem., 72 (2000) 404A].